Understanding protein-protein interactions is important for basic research as well as various biomedical and other practical applications. Examples of this kind include binding between peptide fragments or epitopes and antibodies, the interaction between proteins and short fragments of other proteins, for example, MDM2 and p53 transactivation domain, Bcl-xL and Bak peptide, as well as binding between peptides referred to as aptamers to their target proteins. Development of simple and reliable methods of identifying peptide binders for proteins would help to understand the mechanisms of protein-protein interaction and open new opportunities for drug discovery.
State of the art in silico peptide discovery is guided by the X-ray crystal structures and relies on existing structural information. The application of such methods to de novo discovery of peptide binders is limited. To date, experimental methods provided the most effective approaches for peptide discovery. One approach to identification binders to proteins is the display technology that relies on combinatorial peptide libraries in which peptides are linked to DNA or RNA molecules encoding them. The libraries are panned against immobilized target protein to identify few abundant sequences or so called “winners.” Selection procedure is performed in several rounds. After each round, the sequences of selected peptides are deduced by PCR amplification of the encoding nucleic acid sequences. Different variations of this approach have been developed and successfully applied to peptide discovery; the most commonly used are phage display, ribosome display, and mRNA-display methods. Despite the unquestionable success of these methods at identifying peptide binders, they are expensive, time consuming and prone to contamination. Furthermore, the existing methods do not ensure that the top selected peptide binders are indeed the best and most specific binders and whether they can be improved. First, there is no mechanism that discriminates between specific binders and non-specific ones in the display methods. Second, selecting only a few “winners” prevents display methods from identifying other potentially strong binders that may have had a disadvantage in the selection process. Third, the display methods require careful optimization of the selection conditions for each target protein. Currently, there is no systematic approach that allows selecting an optimal binder for a particular target. Instead, laborious trial and error optimization techniques are used. The present invention addresses this need by providing a systematic approach to fast and reliable discovery of multiple specific binders for a variety of target proteins.
An alternative to display methods to study peptide-protein interactions are peptide arrays. Peptide arrays could be made of peptides synthesized using solid phase peptide synthesis and then immobilized on solid support or could be directly prepared by in situ synthesis methods. Although peptide arrays are commercially available, their application is limited by a relatively low density and high cost of manufacturing. Both of these issues can be addressed by use of maskless light-directed technology, see (Pellois, Zhou et al. (2002) Individually addressable parallel peptide synthesis on microchips) and U.S. Pat. No. 6,375,903. The microarrays are generally synthesized by using light to direct which oligonucleotides or peptides are synthesized at specific locations on an array, these locations being called features. MAS-based microarray synthesis technology allows for the parallel synthesis of millions of unique oligonucleotide or peptide features in a very small area of a standard microscope slide.
Specific peptide binders have multiple applications, including medical diagnostics, drug discovery and biotechnology. The present invention comprises a series of binders to biologically relevant target proteins, identified by an alternative method of fast and reliable discovery of highly-specific peptide binders.